Cardiovascular disease is the leading cause of deaths worldwide. The most common treatments for cardiovascular health diseases are autograft and blood vessel transplantation which has limitations due to lack of donors and the patients' conditions may not allow harvesting one. Additionally, extracting of an autograft may not be possible if all the possible grafts are extracted from the harvested site or the disease has already been advanced. One of the aims of tissue engineering is to provide a possible alternative for such grafts. Recently, tissue engineering and regenerative medicine aim to provide alternative treatments and fast recovery for the patients suffering from cardiovascular diseases. (doi: 10.1155/2012/956345)
Traditionally, tissue engineering strategies are based on the cell seeding into synthetic, biological or composite scaffolds providing a suitable environment for cell attachment, proliferation and differentiation. Cells are seeded into synthetic, biological or composite scaffolds which supply a suitable environment for cell attachment, proliferation and differentiation and have the same functional role as an extracellular matrix (ECM) until the cells create their own ECM. It is really challenging to seed the cells uniformly and selectively so they can attach and proliferate into the fabricated 3D scaffolds. In addition, the seeded scaffolds' degradation could cause immunogenic and unforeseen side effects after in-vivo implantation. In recent years, the scaffolds have been fabricated with controlled internal architecture using 3D printing techniques (doi: 10.1088/1758-5082/3/3/034106, doi: 10.1016/j.cad.2013.07.003).
Although 3D scaffolds are designed to act as an artificial ECM until the cells form their own ECM, it is challenging to fabricate a controlled porous structure with a desired internal architecture repetitively. In addition, functional vascularization of 3D scaffolds is compulsory needed for nutrition and oxygen supply to the engineered tissue. In order to provide nutrition and oxygen to the cells, different approaches based on endothelial cells or scaffolds are developed (doi: 10.1016/j.addr.2011.03.004).
To enable direct anastomosis of the scaffold to the host vasculature in vivo, self-assembly approach is used. In this approach, a bioprinted macrovascular network is matured in a perfusion reactor to achieve required mechanical properties. Microvascular units in the form of cylindrical or spherical multicellular aggregates are produced by the parenchymal and endothelial cells, placed in the macrovascular network and perfused to promote self-assembly and the connection to the existing network (doi: 10.1088/1758-5082/2/2/022001).
Despite several studies related with the vascular tissue engineering, it is still not achieved to construct an entirely biomimetic blood vessel due to the poor mechanical properties of the materials. Therefore, first applications of scaffold-based vascular grafts are tried under low pressure. The degradation of the materials and the cell-material interaction could cause unforeseen side effects including chronic inflammation, thrombosis and rejection after in-vivo implantation. Especially, weakness of cell to cell interaction and the assembly and alignment of ECM are critical in vascular tissue-engineering. Considering all these reasons, vascular tissue engineering studies tend towards scaffold-free techniques (U.S. Pat. No. 8,143,055 B2, US 2012 288 938 A1). In the artificial tissues according to the U.S. Pat. No. 8,143,055 B2, shape and orientation of branches are limited to be parallel with the flat surface on which the cell paste pieces of living cells and their support material pieces are laid. This is a highly binding limitation, which does not fit the natural organization of cells and the shapes of real blood vessels, which have generally uneven shapes and branch orientations.
Additionally, the longitudinal multicellular aggregate preparation method explained in said document requires several repetitive manual bioink preparation steps of multicellular aggregates into/from capillaries; which have to be performed with extreme precision; hence, the reproducibility and speed of said steps can be considered as low. Therefore an alternative method replacing said steps, thus minimizing the human intervention and maximizing the reproducibility is extremely important for bioprinting of said networks.
There has been few research working on building small-diameter, multi-layered, tubular vascular and nerve grafts. Multicellular spherical and cylindrical aggregates have been fabricated with 3D printing methods. Flexibility in tube diameter and wall thickness is obtained and most significantly branched macrovascular structures are constructed with this method (doi:10.1088/1758-5082/4/2/022001). In another study, human embryonic stem cell spheroid aggregates are formed with a valve-based cell printer and they have controllable and repeatable sizes. This work shows that the printed cells are mostly viable and have the potential to differentiate into any of the three germ layers (pluripotency) (doi:10.1088/1758-5082/5/1/015013). However, the preparation of large amounts of spherical aggregates is time-consuming and the fusion process of the spheroids takes 5-7 days. In addition, these approaches mostly require laborious bioink preparation and hence the presented methods can be considered rather unrepeatable and mostly rely on one's own skills.
Valve scaffold tissue engineering has the potential for fabricating blood vessels e.g. aortic valve hydrogel scaffolds that can grow, remodel and integrate with the patient. In order to mimic complex 3D anatomy and heterogeneity of e.g. an aortic valve, root wall and tri-leaflets are 3D printed with poly-ethylene-glycol-diacrylate (PEG-DA) hydrogels. Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds maintained near 100% viability over 21 days (doi: 10.1088/1758-5082/4/3/035005). Another study demonstrates that encapsulated aortic root sinus smooth muscle cells (SMC) and aortic valve leaflet interstitial cells (VIC) are viable within the bioprinted alginate/gelatin aortic valve hydrogel conduits (doi: 10.1002/jbm.a.34420). Recently, human mesenchymal stem cells were encapsulated into agarose hydrogels and cell-laden hydrogel was 3D printed submerged in a hydrophobic high-density fluorocarbon, which mechanically supports the construct and afterwards can be easily removed. This method allows high stability to the printed structures, high cell viability, cell proliferation and production of ECM (doi: 10.1088/1758-5082/5/1/015003). However, the degradation of hydrogel material and formation of tissue structure can take a long time and some of the hydrogel material used could cause immune-reactions or side effects after degradation.
Prior art methods of preparation of multicellular aggregates for bioprinting have limited reproducibility, since said methods require substantial human intervention.
Although there are few studies relevant to the vascular tissue engineering, the poor mechanical strength of the materials contrasted with native vessels has limited the construction of an entirely biomimetic blood vessel. On that account, first implementations of scaffold-based vascular grafts are examined under low pressure (doi: 10.1016/j.biomaterials.2009.06.034).